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RESEARCH COMMUNICATION

␥ differential isoform function. Rationally engineered PPAR knockdown by factors potentially provide a powerful tool engineered transcription for targeted regulation of endogenous by combin- ing a functional transcription regulatory domain with a factors: exogenous PPAR␥2 customized DNA that can bind to a spe- but not PPAR␥1 cific sequence within the target . C2H2 finger (ZFPs) can be engineered to bind with high reactivates adipogenesis specificity to wide a diversity of DNA sequences (Des- jarlais and Berg 1992; Choo and Klug 1994; Jamieson et Delin Ren,1 Trevor N. Collingwood,2 al. 1994; Rebar and Pabo 1994; Greisman and Pabo 1997). Edward J. Rebar,2 Alan P. Wolffe,2 Previous studies have demonstrated the utility of both and Heidi S. Camp1,3 engineered - and repressor-ZFPs in the regula- tion of endogenous chromosomal loci (Bartsevich and 1Department of Molecular Science and Technology, Pfizer Juliano 2000; Beerli et al. 2000; Zhang et al. 2000; Liu et Global and Research Development, Ann Arbor, Michigan al. 2001). Our goal for this study was to selectively in- 48105, USA; 2Sangamo BioSciences Inc., Pt. Richmond, hibit expression of the PPAR␥2 isoform in the adipo- California 94804, USA genic mouse 3T3-L1 cell line by utilizing engineered zinc finger repressor proteins. To determine functional differences between the two splice variants of PPAR␥ (␥1 and ␥2), we sought to se- Results and Discussion ␥ lectively repress 2 expression by targeting engineered The mouse PPAR␥ gene spans >105 kb (Zhu et al. 1995). ␥ zinc finger repressor proteins (ZFPs) to the 2-specific Coding exons 1 to 6 are conserved between the ␥1 and ␥2 promoter, P2. In 3T3-L1 cells, expression of ZFP55 re- isoforms (Fig. 1A) and transcription of these is driven by sulted in >50% reduction in ␥2 expression but had no an upstream promoter (P1) that also drives expression of effect on ␥1, whereas adipogenesis was similarly reduced two untranslated ␥1-specific exons, A1 and A2. The ad- by 50%. However, ZFP54 virtually abolished both ␥2 ditional amino acids at the amino terminus of ␥2 are and ␥1 expression, and completely blocked adipogenesis. encoded by an additional exon, B1, that is uniquely regu- Overexpression of exogenous ␥2 in the ZFP54-expressing lated by a separate promoter (P2) lies >63 kb downstream ␥ cells completely restored adipogenesis, whereas overex- of P1. DNase I digestion of the endogenous PPAR gene locus in 3T3-L1 cells revealed two DNase I hypersensi- pression of ␥1 had no effect. This finding clearly identi- ␥ tive sites in the vicinity of the proximal P2 promoter fies a unique role for the PPAR 2 isoform. that represent regions of accessible at an en- Received October 12, 2001; revised version accepted dogenous locus (Fig. 1B; DHS1 and DHS2). Two six-fin- November 15, 2001. ger ZFPs (ZFP54 and ZFP55; Fig. 1C) linked to the KRAB transcriptional repression domain (Margolin et al. 1994) were designed to bind specifically to 18-bp sequences The nuclear hormone PPAR␥ is essential for within the DHS1 shown in Figure 1B. Each ZFP bound cellular differentiation and accumulation during its cognate site on naked DNA with high affinity adipogenesis (Barak et al. 1999; Kubota et al. 1999; Rosen (Kd = 20 and 44 pM, respectively). et al. 1999). The adipocyte-specific ␥2 isoform differs The ZFP54 and ZFP55 repressor proteins were ex- from the more widely expressed ␥1 in that it contains pressed retrovirally in 3T3-L1 cells to similar levels (Fig. additionally 30 amino acid residues at the amino termi- 2A). Nontransduced wild-type cells and stable pools of nus (Kliewer et al. 1994; Tontonoz et al. 1994a; Zhu et al. infected cells expressing each ZFP, as well as control 1995). Evidence suggests these residues contribute to a cells retrovirally transduced to express LacZ, were in- constitutive transcription activation function that is duced to differentiate and initiate the adipogenic path- 5–10-fold greater than in ␥1 (Werman et al. 1997). way. Total PPAR␥ mRNA level was determined at 0, 2, PPAR␥2 is selectively expressed in adipose tissue (Fajas and 5 d post induction. Over the 5-d time course PPAR␥ et al. 1997) and is strongly up-regulated during adipogen- expression increased ∼5.8- to 6.0-fold in both the wild- esis (Tontonoz et al. 1994b; Wu et al. 1998), suggesting a type and LacZ control cells (Fig. 2B). Up-regulation of specific role for this isoform in fat cell differentiation. total PPAR␥ expression was reduced slightly in the pres- Nevertheless, a specific role for ␥2 that could not be ence of ZFP55 (4.8-fold compared to 6.0-fold in wild type) substituted by ␥1 has not been clearly determined. but completely inhibited by ZFP54. However, a time The ability to selectively knock out or knock down course analysis of expression of the individual PPAR␥ the expression of a specific gene provides a powerful ap- isoforms revealed that although ZFP54 effectively inhib- proach for understanding its biological function. The tar- ited expression of both isoforms, ZFP55 selectively re- geting of individual mRNA splice variants offers an even pressed PPAR␥2by∼50% and had little effect on greater level of selective control and understanding of PPAR␥1 compared with wild-type cells (Fig. 2C). An- other ZFP–KRAB fusion that binds to a sequence that is absent in the PPAR␥ gene has no effect on PPAR␥ [Key Words: PPAR␥1; PPAR␥2; adipogenesis; zinc finger protein] expression or adipogenesis (data not shown). The effects 3 Corresponding author. of these ZFPs are confirmed at the protein level whereby E-MAIL [email protected]; FAX (734) 622-5668. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ PPAR␥2 expression is virtually knocked out in the pres- gad.953802. ence of ZFP54, whereas only a low but detectable level of

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Ren et al.

Figure 1. Structure of PPAR␥ gene and location of accessible chromatin in P2 promoter. (A) Genomic structure of mPPAR␥ showing exon splicing (Zhu et al. 1995). (B) Location of DNase I hypersensitive sites (DHS1 and DHS2) in the proximal P2 pro- moter, along with positions of ZFPs binding sites. (C) ZFP target sequences and finger designs. (Target sequence) The promoter DNA sequence to which each ZFP was designed. (Finger de- signs) The residues in each position from −1 to +6 of the recog- nition helix of each finger targeted against the cognate basepair ␥ triplet subsite. Figure 2. Ectopic expression of PPAR 2-specific ZFPs in 3T3- L1 cells. 3T3-L1 cells were infected with retroviral vectors ex- pressing PPAR␥2 promoter-specific zinc finger repressors, PPAR␥1 remains (Fig. 2D). In the presence of ZFP55 only ZFP54 and ZFP55. The uninfected 3T3-L1 cells (wild type) or PPAR␥2 expression is reduced substantially by ZFP55. cells infected with retroviral vector expressing LacZ gene were Because P1 and P2 are >63 kb apart, it was predicted that used as negative controls. (A) Total RNA was isolated from ZFPs targeted to P2 would selectively inhibit PPAR␥2 selected cell lines and determined the levels of ZFP mRNA by expression. This was the case with ZFP55, which gave Northern blot analysis using a 300 bp C-terminal fragment of 50% knockdown of PPAR␥2 only, however ZFP54 sup- ZFP as probe. The equivalence of RNA loading was verified by pressed both isoforms almost completely. ZFP54 and ␤-actin. (B) Total RNA was isolated on indicated days postdif- ZFP55 bind to opposite strands of the DNA and have ferentiation and subjected to RNase protection assay using a slightly different DNA binding affinities, which may PPAR␥-specific riboprobe. Arrows indicate the protected contribute to their differential effects on P2. PPAR␥ and ␤-actin mRNAs. The fold-induction was calculated The promoter specificity exhibited by the two ZFPs by normalization of PPAR␥-specific signal with ␤-actin signal facilitates examination of the functional requirements and presented as fold-change compared to day-0 of wild-type for each PPAR␥ isoform in adipogenesis. In complete cells. (C) The real time quantitative RT–PCR (TaqMan) analysis concordance with inhibited mRNA and protein expres- of PPAR␥1 and PPAR␥2 mRNA expression. 40 ng and 60 ng of sion, we find that ZFP54 totally blocks the cellular lipid reversed transcribed total RNA was used for PPAR␥2 and accumulation that is a marker of adipogenesis (Fig. 3). PPAR␥1 analysis, respectively. (D) Western blot analysis with a Furthermore, the cells in which only PPAR␥2 is selec- polyclonal antibody against PPAR␥. The arrows indicate tively repressed by 50% (ZFP55) also show a correspond- PPAR␥1 and PPAR␥2 proteins.

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Functional differences between PPAR␥1 and PPAR␥2

also was not inhibited, but rather an increase in its ex- pression was observed in the presence of ZFP54. This suggests a potential negative feedback mechanism whereby PPAR␥ may actively down-regulate C/EBP␦,in keeping with the reduction in C/EBP␦ observed during progression of adipogenesis (Wu et al. 1998). Taken to- gether these results support the notion that the inhibi- tory effects of ZFPs are specific. Genes that are expressed during adipogenesis after PPAR␥ induction include C/EBP␣ and the adipocyte-spe- cific aP2 (Wu et al. 1998; Hamm et al. 2001). As pre- dicted, the loss of PPAR␥ expression in the presence of ZFP54 abrogates both C/EBP␣ and aP2 expression (Fig. 4A,B), supporting a key role for PPAR␥ in the regulatory cascade leading to the expression of these genes. How- ever, in the presence of ZFP55 the expression of C/EBP␣ was delayed (Fig. 4A, lanes 3,7,15), although wild-type expression levels were achieved by day five. This sug- gests that although the remaining PPAR␥ expression in the presence of ZFP55 is sufficient to fully stimulate C/EBP␣ expression, it is not, in itself, sufficient to achieve wild-type levels of cellular differentiation and lipid accumulation. ␥ The studies by others described earlier have demon- Figure 3. Effect of PPAR 2-ZFPs on adipogenesis. Cultured ␥ wild-type 3T3-L1 and retrovirally infected cells were induced to strated an absolute requirement for PPAR expression in differentiate with the standard adipogenic hormones for 10 adipose tissue development, but they failed to differen- tiate clearly between the specific regulatory capacity of days. Cell were fixed in 3% formaldehyde and stained with Oil ␥ ␥ Red O. Stained cells were photographed with Nikon-Di- PPAR 1 and 2 isoforms (Barak et al. 1999; Kubota et al. 1999; Rosen et al. 1999). In the present study, the abro- aphot300 microscope (10 × 20) and 3CCD Vida Camera Systems ␥ (Optronic Engineering). gation of endogenous expression of both PPAR isoforms by ZFP54 provides a unique cell-based model system in which to study the differential functions of each isoform. ing 50% loss in adipogenic capacity, supporting the idea Selective repression of the endogenous PPAR␥ gene of a specific requirement for PPAR␥2 in this pathway. would be expected to generate what otherwise should be A key aspect of engineered ZFPs is the high theoretical an adipogenically competent cell line that is devoid of specificity of DNA binding and gene targeting. Each ZFP is designed to recognize an 18-bp nucleotide sequence that probability dictates to be unique within the mam- malian genome. To further characterize the specificity of PPAR␥ targeting, we analyzed the effect of ZFP expres- sion on other genes both upstream and downstream of PPAR␥ in the adipogenic gene cascade. The cell cyclin- dependent kinase inhibitors p21 and p27 play key roles in cell cycle progression (Harper et al. 1993; Toyoshima and Hunter 1994). P27 is expressed abundantly in growth-arrested preadipocytes where its expression is de- creased transiently on hormonal induction of differentia- tion but returns to initial levels as the cells exit clonal expansion. p21 is absent in growth-arrested cells but in- creases during S-phase of mitotic expansion (Morrison and Farmer 1999). Here we show that the expression pro- file of both p21 and p27 is preserved irrespective of the presence of ZFPs (Fig. 4A). Transient expression of the transcription factors C/EBP␤ and C/EBP␦ occurs very early during adipocyte differentiation in response to the standard adipogenic hormones (IBMX/DEX/Insulin) (Cao et al. 1991; Wu et al. 1998). The proximal promoter of PPAR␥2 contains a tandem array of C/EBP binding elements (Zhu et al. Figure 4. Effects of ZFP54 and ZFP55 on the expression of 1995). PPAR␥ is up-regulated immediately following cyclin-dependent kinase inhibitors, C/EBPs and aP2. Whole C/EBP␤ and C/EBP␦ (Wu et al. 1998) and one would pre- cells extracts and total RNA from the experiment shown in dict that expression of these upstream genes would not Figure 2 were subjected to the following analysis. (A) Western be affected by targeted repression of PPAR␥. In concor- blot analysis of p21 and p27, and C/EBP gene family members dance with this prediction, we found that the expression (␤, ␦, and ␣). Arrows indicate the 42-kD and 30-kD isoforms of level of C/EBP␤ protein was not significantly affected by C/EBP␣.(B) The aP2 gene expression was analyzed by Northern the presence of either ZFPs (Fig. 4A). C/EBP␦ expression blot.

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Ren et al.

only PPAR␥. Rescue of these cells by exogenous expres- sion of either PPAR ␥1or␥2 might be expected to iden- tify isoform-specific functional differences in the capac- ity to potentiate adipogenesis. Here we used retrovirus to overexpress each PPAR␥ isoform on the background of ZFP54 expressing 3T3-L1 cells. Data in Figure 5A,B dem- onstrate the comparable levels of expression of exog- enous PPAR␥1 and ␥2 mRNA and proteins at day 0 in the presence of ZFP54. The exogenously expressed PPAR␥1 isoform was completely incapable of inducing adipogenesis in the presence of ZFP54 (Fig. 5C). In marked contrast, expression of PPAR␥2 effectively re- stored cellular differentiation and lipid accumulation. In addition, PPAR␥2 fully restored C/EBP␣ expression at 7 d postinduction, whereas PPAR␥1 was totally ineffective (Fig. 5D). Neither isoform had any effect on the expres- sion of C/EBP␤ or TBP (Fig. 5D). This result clearly dem- onstrates for the first time a regulatory function for PPAR␥2 in adipogenesis that cannot be achieved by PPAR␥1 in the absence of exogenous . It is unclear how the 30 amino acid residues unique to the PPAR␥2 isoform confer additional regulatory func- tion. However, in addition to rendering the constitutive activation function of the PPAR␥2 amino terminus up to 10-fold greater than that of PPAR␥1(Werman et al. 1997), clinical studies have identified a human allelic variant of at least one of these residues (Pro12Ala) that has been variously associated with decreased receptor activity, lower body mass index, obesity, improved insulin sensi- tivity, and decreased risk of type 2 diabetes (Beamer et al. 1998; Deeb et al. 1998; Altshuler et al. 2000). The effective restoration of the adipogenic cascade by adding back only the product of the targeted gene, PPAR␥, indicates that in this study the use of engineered repressor ZFPs does not result in the general disruption of cellular metabolic processes. This observation, which demonstrates a precise specificity for ZFP targeting, rep- resents an important advance in the rapid establishment of cell-based gene knockdown models and such an ap- proach may, in future, be readily transferable to whole animal transgenic studies.

Materials and methods Design, synthesis, and DNA binding affinity of zinc finger proteins Pairs of 3-finger ZFPs were generated as described previously (Zhang et al. 2000) then linked to form each 6-finger ZFP. The DNA binding affin- ity of each ZFP was determined using a gel shift technique essentially as described earlier (Zhang et al. 2000) except that 10 µM zinc was used.

Plasmids and stable cell lines ␥ ␥ The retroviral expression vector pBMN (Garry Nolan Laboratory, Stan- Figure 5. Ectopic expression of PPAR 2, but not 1 restores ford University) was constructed by inserting an internal ribosome entry adipogenesis in ZFP54 cells. The L1/ZFP54 cells were infected site (IRES) along with either puror or neor. The retroviral plasmids pBM- with the retroviral constructs expressing either PPAR␥1 Npuro–ZFP54 and pBMNpuro–ZFP55 were constructed by inserting (pBMN/neo-␥1) or PPAR␥2 (pBMN/neo-␥2). The retroviral vec- ZFP54 and ZFP55 cDNA fragments into pBMN–puro vector at EcoRI and tor expressing LacZ was used as empty vector control. The dou- XhoI sites. pBMN/neo-␥1 was generated by inserting the full-length mP- bly infected cell lines were designated as ZFP54/␥1, ZFP54/␥2, PAR␥1 cDNA (nucleotide positions −6 to +1415) into pBMN–neo vector and ZFP54/LacZ, respectively. After the selection with puro- at BamHI and NotI sites. pBMN/neo-␥2 construct was derived from mP- ␥ mycin and geneticin, cells were subjected to the differentiation PAR 2/pSport (a gift from Dr. B. Spiegelman, Harvard Medical School, Boston, MA) by specifically ligating the blunt-ended PPAR␥2 cDNA into protocol as described previously. (A) Total RNA was isolated ␥ pBMN/neo vector at the blunt-ended EcoRI site. To generate infectious from the indicated cell lines and PPAR mRNA level was quan- recombinant viruses, Phoenix-ECO cells (Garry Nolan Laboratory, Stan- ␥ ␥ titated by RNase protection assay. (B) PPAR 1 and 2 protein ford University, CA) were cultured in DMEM medium containing 10% levels by Western blot analysis. (C) Cells were differentiated FBS. Cells were transfected with 15 µg of retroviral plasmids by Lipo- with the adipogenic hormones for 10 d and stained with Oil Red fectamine 2000 kit (GIBCO BRL) following the protocol as recommended O. (D) C/EBP␣ and ␤ protein expression by Western blot analy- by the manufacturer. Viral supernatant was harvested at 72 h posttrans- sis. The membrane was stripped and reblotted with a rabbit fection and added onto ∼75% confluent 3T3-L1 cells in the presence of 8 polyclonal antibody against TBP. µg/mL polybrene (Sigma) for 2 h. Infected cells were selected by adding 2

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µg/mL puromycin (Sigma) for 5 d. For double infection, L1/ZFP54 cells ies. Mol. Pharmacol. 58: 1–10. were infected with either pBMN/neo-␥1, or pBMN/neo-␥2, and cells were Beamer, B.A., Yen, C.J., Andersen, R.E., Muller, D., Elahi, D., Cheskin, selected with both 600 µg/mL geneticin (GIBCO BRL) and 2 µg/mL pu- L.J., Andres, R., Roth, J., and Shuldiner, A.R. 1998. Association of the romycin for 14 d. Pools of stably infected cells were used in these ex- Pro12Ala variant in the peroxisome proliferator-activated receptor-␥2 periments. gene with obesity in two Caucasian populations. Diabetes 47: 1806– 1808. Cell culture and differentiation Beerli, R.R., Dreier, B., and Barbas, C.F., III. 2000. Positive and negative Uninfected 3T3-L1 and virally infected 3T3-L1 cells were cultured and regulation of endogenous genes by designed transcription factors. maintained for 2 d postconfluence in DMEM containing 10% CS. Dif- Proc. Natl. Acad. Sci. 97: 1495–1500. ferentiation protocol was conducted as described previously (Camp et al. Camp, H.S., Whitton, A.L., and Tafuri, S.R. 1999. PPAR␥ activators 2001) down-regulate the expression of PPAR␥ in 3T3-L1 adipocytes. FEBS Lett. 447: 186–190. Mapping of DNase I accessible chromatin regions Camp, H.S., Chaudhry, A., and Leff, T. 2001. A novel potent antagonist DNase I mapping was performed essentially as described previously (Liu of peroxisome proliferator-activated receptor gamma blocks adipo- et al. 2001). Undifferentiated 3T3-L1 cell nuclei were partially digested cyte differentiation but does not revert the phenotype of terminally with DNase I and the genomic DNA was extracted followed by XbaI differentiated adipocytes. Endocrinology 142: 3207–3213. digestion at −1588 bp in the P2 promoter. The probe for Southern blotting Cao, Z., Umek, R.M., and McKnight, S.L. 1991. Regulated expression of of the proximal P2 promoter was an XbaI/EcoRI fragment spanning from three C/EBP isoforms during adipose conversion of 3T3-L1 cells. −1588 to −1007. Genes & Dev. 5: 1538–1552. Choo, Y. and Klug, A. 1994. Selection of DNA binding sites for zinc The real-time quantitative PCR (TaqMan) fingers using rationally randomized DNA reveals coded interactions. Gene-specific primers and probes were designed using the Primer Express Proc. Natl. Acad. Sci. 91: 11168–11172. software (Perkin Elmer Life Sciences). The real-time quantitative RT– Deeb, S.S., Fajas, L., Nemoto, M., Pihlajamaki, J., Mykkanen, L., Kuu- PCR reaction was performed essentially following the manufacturer’s sisto, J., Laakso, M., Fujimoto, W., and Auwerx, J. 1998. A Pro12Ala protocol. Briefly, reaction mixture contained 5.5 mM MgCl2, 500 µM substitution in PPAR␥2 associated with decreased receptor activity, dNTP, 2.5 µM random hexamers, 200 nM FAM-probe, and 600 nM of lower body mass index and improved insulin sensitivity. Nat. Genet. both forward and reverse primers in a final volume of 25 µL, and was 20: 284–287. analyzed in ABI PRISM 7700 sequence detection system (Perkin Elmer Desjarlais, J.R. and Berg, J.M. 1992. Redesigning the DNA-binding speci- Life Sciences). Relative quantitation of PPAR␥ mRNA levels was plotted ficity of a zinc finger protein: A data base-guided approach. Proteins as fold-change compared to day 0 of wild-type 3T3-L1 cells. 18S ribo- 12: 101–104. somal RNA was used for normalization. TaqMan reverse transcriptase Fajas, L., Auboeuf, D., Raspe, E., Schoonjans, K., Lefebvre, A.M., Saladin, reactions were performed in triplicates and the experiments were re- R., Najib, J., Laville, M., Fruchart, J.C., Deeb, S., et al. 1997. The peated independently at least three times. organization, promoter analysis, and expression of the human PPAR␥ gene. J. Biol. Chem. 272: 18779–18789. RNA isolation, Northern blot, and RNase protection assay Greisman, H.A. and Pabo, C.O. 1997. A general strategy for selecting Total RNA was isolated from cultured cells using Ultraspec RNA system high-affinity zinc finger proteins for diverse DNA target sites. Sci- (Biotecx Laboratories, Inc.). ZFP, aP2, and rat ␤-actin cDNA were labeled ence 275: 657–661. with [␣-32P] dCTP (Amersham) using random primed labeling kit Hamm, J.K., Park, B.H., and Farmer, S.R. 2001. A role for C/EBP␤ in (GIBCO BRL). Northern blot analysis and RNase protection assay (RPA) regulating peroxisome proliferator-activated receptor gamma activity were performed as described previously (Camp et al. 1999). during adipogenesis in 3T3-L1 preadipocytes. J. Biol. Chem. 276: 18464–18471. Immunoblot analysis Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K., and Elledge, S.J. 1993. Cells were lysed in HNTG cell lysate buffer (50 mM Hepes, 150 mM The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 NaCl, 10% glycerol, 1% Triton, 1.5 mM MgCl2, and 1 mM EDTA) at 4°C cyclin-dependent kinases. Cell 75: 805–816. for 15 min, followed by centrifugation at 15,000 rpm at 4°C for 10 min. Jamieson, A.C., Kim, S.H., and Wells, J.A. 1994. In vitro selection of zinc The supernatant was collected and the protein concentration was deter- fingers with altered DNA-binding specificity. Biochemistry 33: 5689– mined by BCA protein assay (Pierce). Western blot analysis was carried 5695. out as described previously (Camp et al. 1999). Rabbit polyclonal anti- Kliewer, S.A., Forman, B.M., Blumberg, B., Ong, E.S., Borgmeyer, U., bodies against C/EBP␣, C/EBP␤, C/EBP␦, TBP, and monoclonal antibod- Mangelsdorf, D.J., Umesono, K., and Evans, R.M. 1994. Differential ies against p21 and p27 were purchased from Santa Cruz. expression and activation of a family of murine peroxisome prolif- erator-activated receptors. Proc. Natl. Acad. Sci. 91: 7355–7359. Acknowledgments Kubota, N., Terauchi, Y., Miki, H., Tamemoto, H., Yamauchi, T., Kom- eda, K., Satoh, S., Nakano, R., Ishii, C., Sugiyama, T., et al. 1999. We thank Neelam Srivastava, Katherine Pasquetti, and Hong Qi for tech- PPAR␥ mediates high-fat diet-induced adipocyte hypertrophy and in- nical assistance, and Casey Case, Todd Leff, and Yen Choo for valuable sulin resistance. Mol. Cell 4: 597–609. discussion and criticism. We dedicate this work to the memory of our Liu, P.Q., Rebar, E.J., Zhang, L., Liu, Q., Jamieson, A.C., Liang, Y., Qi, H., friend and colleague, Alan P. Wolffe. Li, P.X., Chen, B., Mendel, M.C., et al. 2001. Regulation of an endog- The publication costs of this article were defrayed in part by payment enous locus using a panel of designed zinc finger proteins targeted to of page charges. This article must therefore be hereby marked “adver- accessible chromatin regions. Activation of vascular endothelial tisement” in accordance with 18 USC section 1734 solely to indicate this growth factor A. J. Biol. Chem. 276: 11323–11334. fact. Margolin, J.F., Friedman, J.R., Meyer, W.K., Vissing, H., Thiesen, H.J., and Rauscher, F.J., III. 1994. Kruppel-associated boxes are potent tran- References scriptional repression domains. Proc. Natl. Acad. Sci. 91: 4509–4513. Morrison, R.F. and Farmer, S.R. 1999. Role of PPAR␥ in regulating a Altshuler, D., Hirschhorn, J.N., Klannemark, M., Lindgren, C.M., Vohl, cascade expression of cyclin-dependent kinase inhibitors, p18(INK4c) M.C., Nemesh, J., Lane, C.R., Schaffner, S.F., Bolk, S., Brewer, C., et and p21(Waf1/Cip1), during adipogenesis. J. Biol. 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PPARγ knockdown by engineered transcription factors: exogenous PPAR γ2 but not PPARγ1 reactivates adipogenesis

Delin Ren, Trevor N. Collingwood, Edward J. Rebar, et al.

Genes Dev. 2002, 16: Access the most recent version at doi:10.1101/gad.953802

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